One of the mainstays of glaucoma treatment is the use of drugs that decrease the secretion of aqueous humor fluid from the ciliary epithelium. Unfortunately, many currently available drugs that decrease aqueous humor production such as .beta.-adrenergic antagonists, may cause serious systemic side effects such as cardiac arrhythmias, and arrest, pulmonary bronchospasm, and CNS side effects such as decreased libido and depression [13, 40, 66]. Similarly, the systemic use of carbonic anhydrase inhibitors is associated with blood dyscrasias, renal calculi, and mental changes such as confusion and depression, paresthesia of the extremities and electrolyte disturbances [12].
The development of effective aqueous suppressants in patients who are suffering from glaucoma will result in decreased morbidity and mortality in the population. Glaucoma is a complex disease characterized chiefly by an increase in intraocular pressure. If the intraocular pressure is sufficiently high and persistent, it may lead to damage to the optic disc at the juncture of the optic nerve and the retina. The result of high intraocular pressure is irreversible and can cause blindness. There are three types of glaucoma characterized: primary, secondary, and congenital.
The vacuolar H.sup.+ -ATPase (V-ATPase) resides on the plasma membrane of the ciliary epithelium and acts-as an important ion motive force in aqueous humor production. Translocation of this enzyme to and from the plasma membrane appears to be an important mechanism by which aqueous humor production may be regulated.
No model exists to explain the action of carbonic anhydrase inhibitors in the ciliary epithelium in the non-pigmented epithelium (NPE). Immunostaining of the NPE in rabbit ciliary epithelium was identified [9]. As described herein, we disclose that a proton pump belonging to a different class of enzymes, namely the vacuolar H.sup.+ -ATPases, is present in the basolateral membrane of the nonpigmented and pigmented epithelium (PE) cells of the rabbit ciliary epithelium and in SV-40 transformed human-derived nonpigmented and bovine-derived pigmented ciliary epithelial cells grown in tissue culture.
The V-ATPase pump is recruited to the plasma membrane in the ciliary epithelium and in cultured ciliary epithelial cells by drugs which alter adenylyl cyclase and phospholipase C. Drugs which alter these second messenger systems are thought to be important in the regulation of aqueous humor production [68]. Furthermore, drugs such as propranolol, a .beta.-adrenergic antagonist, inhibited the recruitment of proton ATPase to the plasma membrane.
Most of the membrane organelles in a typical eukaryotic cell belong to the elements of the exocytic and endocytic pathways, referred to collectively as the vacuolar system [37]. They include the endoplasmic reticulum, the golgi complex, the secretory vacuoles, the endosomes, the lysosomes, and other organelles involved in biosynthesis, processing, transport, storage, release, and degradation of soluble and membrane-bound macromolecules. An important similarity among the organelles of the vacuolar system is the presence of H.sup.+ -ATPases responsible for generating all internal acidic environment.
There are two mechanistically distinct groups of ATP dependent ion pumps. One group, the P-ATPases (E.sub.1 E.sub.2 ATPases) operate with a phosphoenzyme intermediate and its members (i.e. Na.sup.+ /K.sup.+ ATPase, gastric H.sup.+ ATPase) are usually sensitive to low concentration of vanadate, a phosphate transition state analog. The P-ATPases are present in the cell membranes of fungi, plants, animals, sarcoplasmic reticulum of muscle cells, and the bacterial cytoplasmic membranes.
The other group, contains the families of F.sub.1 F.sub.0 ATPases (F-ATPases) and vacuolar H.sup.+ -ATPases (V-ATPases) [21, 39, 42, 43]. F-ATPases and V-ATPases function without a phosphorylated intermediate, [41] are multisubunit protein complexes that are built of distinct catalytic and membrane sectors, are not sensitive to low vanadate concentrations, but are sensitive to bafilomycin A [5, 70, 77].
The vacuolar proton ATPases are distinguished from the other two classes by virtue of their inhibitor specificities, lack of coupling to counter-ion transport, and intracellular distribution. V-ATPase inhibitors include: N-ethylmaleimide (NEM), 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) [3, 16, 18, 49, 67] N,N'-dicyclohexylcarbodiimide, Nethymaleimide, NO.sub.3 -, bafilomycin A [5, 70], concanamycin [77], suramin [38] and fusidic acid [38].
The family of V-ATPases are present in archaebacteria and vacuolar systems of eukaryotic cells. While the family of F-ATPases function in eubacteria and are present exclusively in the thylakoid membrane of chloroplasts, inner mitochondrial membrane and bacterial cytoplasmic membranes.
V-ATPases pump protons without internal counterions and therefore are inherently electrogenic. Since they use ATP, they are also strongly oxygen-dependent in cells with low anaerobic phosphorylating capacity. They energize membranes by transducing the energy from ATP hydrolysis into a proton current, which establishes an electrochemical gradient .increment..mu.H.
The vacuolar H.sup.+ -ATPases are ubiquitous intracellular proton pumps in cells. They serve to acidify intracellular compartments and organelles in mammalian cells such as lysozomes, golgi, and synaptic vesicles and thus lower intralumenal pH. In addition, the vacuolar H.sup.+ -ATPases may serve to modulate cellular functions in conjunction with its external milieu.
The vacuolar H.sup.+ -ATPases are all 500 kD-600 kD molecular weight (Mr) proteins with generally at least eight different component subunits. All have subunits of approximately 70 kD, 56 kD, several subunits between 30 and 50 kD, and at least one low molecular weight subunit of 17 kD. In all of the enzymes, most of the large molecular weight proteins are peripheral membrane proteins that do not have any membrane spanning portions, and the small molecular weight proteins are intrinsic membrane proteins that span the lipid bilayer.
The vacuolar H.sup.+ -ATPases have two major domains, the cytoplasmic domain and the transmembrane domain (FIG. 1). The cytoplasmic domain is the locus of the catalytic and probable regulatory sites of the enzyme, and is composed of peripheral membrane proteins. The transmembrane domain forms the channel through which protons cross the lipid bilayer and is composed of intrinsic membrane proteins which the cytoplasmic domain and anchor it on the membrane.
The cytoplasmic domain contains the .about.70 kD ("A") subunit of the vacuolar H.sup.+ -ATPases and appear to have the site where ATP is hydrolyzed during proton transport. There are 3 subunits of 70 kD in each complete proton pump. Cloning and Southern blotting of this subunit from bovine genomic DNA suggests that there is only one gene for the 73 kD subunit [49]. The sequences of the 70 kD subunit and its homolog have a highly conserved domain in the mid-coding region which comprise the nucleotide binding and catalytic site. The sequence of the bovine kidney subunit compared with the plant and the fungal enzymes show wide divergence at the amino-terminal and carboxyl-terminal domains, with no sequence conservation. It is possible that these regions have a regulatory or non-catalytic role.
There are 3 subunits of 56 kD ("b" subunit) per H.sup.+ -ATPase. The function of this subunit is uncertain. The subunit is homologous to the .alpha.subunit of the F.sub.0 F.sub.1 H.sup.+ -ATPase. The .alpha.subunit does not have a catalytic ATP binding site, but is required for catalytic activity. It has a high affinity nucleotide binding site which is thought to be involved either in regulation of the enzyme, or as a non-hydrolytic part of the reaction mechanism. The function of the 56 kD subunit of the vacuolar H.sup.+ -ATPases ATPases is unknown although evidence from the plant enzyme suggests that it may have an ATP binding site. Cloning of the subunit has revealed that there are at least two different isoforms of the 56 kD subunit in the kidney, and these are encoded by different genes [44, 50].
Vacuolar H.sup.+ -ATPase affinity purified from a bovine kidney cortex microsomal fraction had different enzymatic properties from V-ATPase isolated from bovine kidney brush border, in addition to heterogeneity of the 56 kD and 31 kD subunits [67]. The 56 kD subunit therefore has an important role in determining the tissue specific enzymatic properties or compartmentation of the vacuolar H.sup.+ -ATPase.
There are from 1 to 3 subunits of the 31 kD per H.sup.+ -ATPase. The cDNA has been cloned from bovine kidney [35]. Monoclonal antibodies raised against heterogeneous 31 kD subunits in the renal brush border and collecting tubules are consistent with preliminary data from immunoscreening genomic DNA that suggests more than 1 gene codes for this subunit [24]. The amino acid sequence of the subunit is 98% identical between different mammalian species, far higher than for the 70 and 56 kD subunits.
The function of the other subunits of the cytosolic domain is not established, but they may constitute a "stalk" domain connecting the catalytic portion to the intrinsic membrane domain similar to the construction of the F.sub.0 F.sub.1 enzymes.
The transmembrane domain forms a proton conducting channel that spans the lipid bilayer [41]. Although the entire composition of this portion of the enzyme remains in dispute, all of the vacuolar H.sup.+ -ATPases have an approximately 17 kD (or 15 kD in the kidney) polypeptide that reacts readily with the hydrophobic .alpha.-carboxyl reagent dicyclohexylcarbodiimide.
The ciliary epithelium is a double layer epithelium composed of two cell types whose apical ends face each other. Both the outer nonpigmented (NPE) and inner pigmented (PE) epithelial layers exhibit properties of transporting epithelia [23]. The NPE is thought to provide the direct driving force for aqueous humor formation. Physiologic and immunocytochemical evidence suggests that the Na.sup.+ /K.sup.+ -ATPase resides in the basolateral membrane driving sodium secretion and providing the main ion motive force for driving sodium dependent cotransporters [7, 11, 15, 45, 65].
Electroneutrality is thought to be maintained by anion channels in the NPE basolateral membrane. However, the NPE is coupled to the PE through an extensive network of gap junctions and therefore the bilayer is thought to function electrogenically as a syncytium [32, 55, 71, 72]. Solute entry in to dual epithelium is thought to occur at the basolateral surface of the pigmented epithelial cells through several sodium dependent cotransporters (Na.sup.+ -H exchange, Na.sup.+ dependent NaHCO.sub.3 - exchange, electroneutral Na.sup.+ Cl.sup.- cotransport and others) [6, 73, 76].